Hitoshi Shiku

Rapid immunosensing based on accumulation of microparticles by negative dielectrophoresis

In the work, microfluidic device consisting of an interdigitated microarray (IDA) electrode was developed for a rapid, and separation-free immuno-sensors based on a manipulation technique of microparticles by dielectrophoresis (DEP). A poly-dimethylsiloxane (PDMS) substrate with microfluidic channel was placed on the IDA plate to allow to fabricating the device. On applying AC voltage to the IDA in a negative DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microparticles moved to the surface of PDMS substrate placed above the IDA by n-DEP force to accumulate at the designated areas of the PDMS surface, where anti-mouse IgG was precoated. When the fluorescence microparticles bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microparticles trapped the analyte to form microparticle-conjugates. The conjugates were accumulated and captured at the designated areas of the PDMS surface via antibody-antigen-antibody (sandwich) reaction. The captured microparticles were detected selectively by fluorescence measurements at the focused, designated areas regardless of the presence of uncaptured microparticles in the suspended solution. Thus, the separation and washing-out steps, usually required for conventional immunoassay, are eliminated in the presented procedure. Since the formation of the sandwich structures was accelerated significantly by n-DEP, as short as 30 sec was enough to detect the immunoreaction at the surface. The fluorescence intensity of the captured microparticles at the designated area increased with the analyte in the range, 0.01 ~ 10 ng/mL. The present procedure realizes a rapid, sensitive and separation-free immunoassay in a simple device.

Negative dielectrophoretic manipulation with microparticles for rapid immunosensing

Negative dielectrophoresis (n-DEP) have been used to manipulate microparticles with immunoreagents (antigens or antibodies) in a microfluidic channel, and applied to develop a rapid immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 mum diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area by using n-DEP. A sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing FITC-labeled anti-mouse IgG antibody, into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 mug/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. This immunosensing system using the n-DEP technique is faster and simpler than conventional enzyme-linked immunosorbent assay (ELISA) using microtiter plates, and has the significant advantage that sensing requires simple and easy handling since unreacted immunomolecules are flushed from the signal detection area by the fluidic stream. The device can be reused by removing the microparticles. The automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP.